Assay devices with integrated sample dilution and dilution verification and methods of using same

ABSTRACT

The invention is to devices and method for rapid determination of analytes in liquid samples by various assays including immunoassays incorporating a sample dilution feature for forming a diluted sample for analysis. The devices and methods also include a dilution verification feature for verifying the degree of dilution of the diluted sample. The devices preferably are capable of being used in the point-of-care diagnostic field is provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 13/308,968, filed on Dec. 1, 2011, which claims priority toU.S. Provisional Application No. 61/419,505, filed Dec. 3, 2010, theentire contents and disclosures of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention generally relates to devices and methods for rapiddetermination of analytes in liquid samples by various assay techniquesincluding immunoassays incorporating a sample dilution feature, whichmay be configured for high range dilution or low range dilution. Thedevices and methods desirably have the ability to verify the degree ofdilution, preferably by using a dilution determinant marker. Theapparatus preferably is capable of being used in the point-of-carediagnostic field, including, for example, use at accident sites,emergency rooms, in surgery, in intensive care units, and also innon-medical environments.

BACKGROUND OF THE INVENTION

A multitude of laboratory immunoassay tests for analytes of interest areperformed on biological samples for diagnosis, screening, diseasestaging, forensic analysis, pregnancy testing and drug testing, amongothers. While a few qualitative tests, such as pregnancy tests, havebeen reduced to simple kits for a patient's home use, the majority ofquantitative tests still require the expertise of trained technicians ina laboratory setting using sophisticated instruments. Laboratory testingincreases the cost of analysis and delays the patient's receipt of theresults. In many circumstances, this delay can be detrimental to thepatient's condition or prognosis, such as for example the analysis ofmarkers indicating myocardial infarction and heart failure. In these andsimilar critical situations, it is advantageous to perform such analysesat the point-of-care, accurately, inexpensively and with minimal delay.

Many types of immunoassay devices and processes have been described. Forexample, a disposable sensing device for measuring analytes by means ofimmunoassay in blood is disclosed by Davis et al. in U.S. Pat. No.7,419,821, the entirety of which is incorporated herein by reference.This device employs a reading apparatus and a cartridge that fits intothe reading apparatus for the purpose of measuring analyteconcentrations. A potential problem with such disposable devices isvariability of fluid test parameters from cartridge to cartridge due tomanufacturing tolerances or machine wear. U.S. Pat. No. 5,821,399 toZelin, the entirety of which is incorporated herein by reference,discloses methods to overcome this problem using automatic flowcompensation controlled by a reading apparatus having conductimetricsensors located within a cartridge.

Electrochemical detection, in which the binding of an analyte directlyor indirectly causes a change in the activity of an electroactivespecies adjacent to an electrode, has also been applied to immunoassays.For an early review of electrochemical immunoassays, see Laurell et al.,Methods in Enzymology, vol. 73, “Electroimmunoassay”, Academic Press,New York, 339, 340, 346-348 (1981).

In an electrochemical immunosensor, the binding of an analyte to itscognate antibody produces a change in the activity of an electroactivespecies at an electrode that is poised at a suitable electrochemicalpotential to cause oxidation or reduction of the electroactive species.There are many arrangements for meeting these conditions. For example,electroactive species may be attached directly to an analyte, or theantibody may be covalently attached to an enzyme that either produces anelectroactive species from an electroinactive substrate or destroys anelectroactive substrate. See M. J. Green (1987) Philos. Trans. R. Soc.Lond. B. Biol. Sci. 316:135-142, for a review of electrochemicalimmunosensors. Magnetic components have been integrated withelectrochemical immunoassays. See, for example, U.S. Pat. Nos.4,945,045; 4,978,610; and 5,149,630, each to Forrest et al. Furthermore,jointly-owned U.S. Pat. No. 7,419,821 to Davis et al. (referenced above)and U.S. Pat. Nos. 7,682,833 and 7,723,099 to Miller et al. teachelectrochemical immunosensing devices and methods.

Microfabrication techniques (e.g., photolithography and plasmadeposition) are attractive for construction of multilayered sensorstructures in confined spaces. Methods for microfabrication ofelectrochemical immunosensors, for example on silicon substrates, aredisclosed in U.S. Pat. No. 5,200,051 to Cozette et al., the entirety ofwhich is incorporated herein by reference. These include dispensingmethods, methods for attaching biological reagent, e.g., antibodies, tosurfaces including photoformed layers and microparticle latexes, andmethods for performing electrochemical assays.

In U.S. Pat. No. 4,946,795, Gibbons et al. disclose a sample dilutioncartridge that relies on hydrostatic pressure. Jointly-owned U.S. Pat.No. 6,750,053 to Widrig et al., the entirety of which is incorporatedherein by reference, teaches sample metering based on a holding chamberwith a capillary stop feature.

Notwithstanding the above literature, there remains a need in the artfor improved immunosensing devices with a greater range of detection ofanalytes, including, for example, analytes present at low levels such ascardiac troponin I, and analytes present at high levels, such as CRP.The need also exists for improved devices and methods for meteringsamples, particularly in point-of-care analyte testing. These and otherneeds are met by the present invention as will become clear to one ofskill in the art to which the invention pertains upon reading thefollowing disclosure.

SUMMARY OF THE INVENTION

The present invention is directed to immunosensing devices and methodsof performing an immunoassay with immunosensors incorporating a sampledilution feature and a dilution verification feature for verifying thedegree of dilution of the diluted sample. In some embodiments, thedevices and methods are configured for high range dilutions, e.g.,greater than about 50:1, while in other embodiments, the devices andmethods are configured for low range dilution, e.g., dilutions that areless than about 50:1 (v/v diluent:sample). The devices preferablyprovide diverse real-time or near real-time analysis of analytes.

In one embodiment, the invention is to a method of performing an assayfor an analyte in a fluid sample with a cartridge having an integratedsample dilution element, where the cartridge is adapted for insertioninto a reading apparatus, the method comprising: (a) introducing a fluidsample into a sample holding chamber of a cartridge with a sampledilution element, wherein at least a portion of said dilution elementdetermines a volume of sample for dilution, and wherein said dilutionelement further comprises a predetermined known amount of a dilutiondeterminant marker capable of dissolving into said sample; (b) pumping ametered volume of diluent from a diluent chamber in said cartridge, tosaid sample dilution element to form a diluted sample; (c) pumping thediluted sample to a sensor in a sensing region of said cartridge; (d)measuring the concentration of said dilution determinant marker in saiddiluted sample; and (e) determining the dilution ratio of the dilutedsample from said measured concentration in step (d) and saidpredetermined known amount of step (a). Depending on how the cartridgeis configured, the diluted sample may be at a dilution ratio of fromabout 1:1 to 50:1 parts by volume diluent:sample, or from 50:1 to about50,000:1. The predetermined known amount of dilution determinant markermay be an embedded value in said reading apparatus, an embedded value onsaid assay cartridge and automatically read by said reading apparatus, avisible value on said assay cartridge that is manually entered into saidreading apparatus, or a visible value on said assay cartridge packagethat is manually entered into said reading apparatus. The dilution ratiovalue determined in step (e) preferably is used to calculate theconcentration of the analyte in the undiluted sample.

In another embodiment, the invention is to a sensor cartridge forsensing at least one analyte in a sample, comprising: at least onesensor in a analysis conduit; a sample chamber between a sample entryport and a sample extraction unit, wherein said extraction unitcomprises a dilution determinant marker; a diluent package containing adiluent; a diluent conduit configured for transporting diluent from thediluent package to the sample extraction unit; and a pump configured totransfer said diluent through said diluent conduit, over and/or throughsaid sample extraction unit, and into said analysis conduit. The diluentconduit preferably defines a metered volume of diluent. The cartridge isparticularly well suited for diluting said sample at a dilution ratio ofabout 50:1 to about 50,000:1 (v/v diluent:sample).

In another embodiment, the invention is to a method of performing anassay for an analyte in a sample, said method comprising the steps of:introducing a sample into a sample chamber of a cartridge, wherein thesample chamber terminates in a sample extraction unit comprising adilution determinant marker; loading said extraction unit with saidsample; washing a portion of said sample from the extraction unit usinga volume of diluent from a diluent conduit to form a diluted sample;measuring the amount of dilution determinant marker in said dilutedsample; and analyzing said diluted sample for an analyte. The methodpreferably further comprises the step of calculating a dilution ratio ofsaid diluted sample.

In another embodiment, the invention is to a method of performing anassay for an analyte in a sample, said method comprising the steps of:adding a dilution determinant marker to the sample; measuring thedilution determinant marker concentration in the sample; introducing thesample into a sample chamber of a cartridge, wherein the sample chamberterminates in a sample extraction unit; loading said extraction unitwith said sample; washing a portion of said sample from the extractionunit using a volume of diluent from a diluent conduit to form a dilutedsample; measuring the dilution determinant marker concentration in saiddiluted sample; comparing the dilution determinant marker concentrationin the sample prior to introducing said sample into the sample chamberwith the dilution determinant marker concentration in the portion of thesample washed from the extraction unit; and analyzing said dilutedsample for an analyte.

In another embodiment, the invention is to a method of performing anassay for an analyte in a sample, said method comprising the steps of:introducing a sample into a sample chamber of a cartridge, wherein thesample chamber terminates in a sample extraction unit, which preferablycomprises a dilution determinant marker used in characterizing thedilution ratio; loading said extraction unit with said sample; washing aportion of said sample from the extraction unit using a volume ofdiluent from a diluent conduit to form a diluted sample; analyzing saiddiluted sample for an analyte; and characterizing a dilution ratio ofthe diluted sample. The method preferably further comprises the steps oftransporting said diluted sample to a sensor; and performing an analyteassay at said sensor. The sample preferably is diluted in the range ofabout 50:1 to about 50,000:1 (v/v diluent:sample).

Dilution verification may also be employed in low range dilutionembodiments, e.g., embodiments in which the diluted sample is at adilution ratio of from 1:1 to 50:1 parts by volume diluent:sample. Forexample, in one embodiment, the invention is to a method of performingan assay for an analyte in a sample, said method comprising the stepsof: introducing a sample into a sample chamber of a cartridge, whereinsaid chamber is located between a sample entry port and a sampleisolation unit, preferably a capillary stop, and includes a diluentintroduction port for receiving diluent, and wherein the volume betweenthe diluent introduction port and the sample isolation unit defines ametered volume of said sample for dilution; transporting a volume ofdiluent from a diluent conduit to the metered volume of sample, whereinsaid volume of diluent is combined with said metered volume of sample toform a diluted sample; transporting said diluted sample to a sensor;performing an analyte assay at said sensor; and characterizing adilution ratio of the diluted sample. The method preferably furthercomprises the step of adding a dilution determinant marker to saidsample, wherein said dilution ratio is characterized using the dilutiondeterminant marker. The volume of diluent optionally comprises a meteredvolume of diluent. The cartridge preferably includes a pump conduitbeginning at a pump and ending at an air introduction port on thediluent conduit, and wherein a region in the diluent conduit between theair introduction port and the diluent introduction port defines themetered volume of diluent. In some embodiments, the sample isolationunit may be selected from the group consisting of a porous hydrophilicmaterial, a cellulose material, nitrocellulose, cotton fiber, paper, andglass-filled paper.

In the above embodiments, the dilution determinant marker may, forexample, be selected from the group consisting of an electrochemicalspecies, ferricyanide, ruthenium hexamine, a ferrocene, ferrocenemonocarboxylic acid, an optical dye, fluorescein, an acridinium salt,and methylene blue and the like. The dilution determinant markerconcentration may, for example, be measured using an electrochemicalanalysis system for amperometric measurements, using a potentiometricsensor, or using a spectrophotometer.

The sensor(s) employed in the devices of the invention preferably areselected from the group consisting of an immunosensor, an ion sensor, ametabolite sensor, an enzymatic sensor, an enzyme activity sensor and anucleotide sensor. In each embodiment, the sample may be selected, forexample, from the group consisting of blood, plasma, serum, urine,interstitial fluid and cerebrospinal fluid.

The above summary of the present invention is not intended to describeeach illustrated embodiment or every implementation of the presentinvention. The figures and the detailed description that follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objectives, features and advantages of the presentinvention are described in the following detailed description of thespecific embodiments and are illustrated in the following Figures, inwhich:

FIGS. 1A and 1B are isometric top and bottom views, respectively, of animmunosensor cartridge cover in accordance with one embodiment of thepresent invention;

FIG. 2 is a top view of the layout of a tape gasket for an immunosensorcartridge in accordance with one embodiment of the present invention;

FIG. 3 is an isometric top view of an immunosensor cartridge base inaccordance with one embodiment of the present invention;

FIG. 4 is an exploded view of an immunosensor cartridge according to oneembodiment of the invention;

FIG. 5 is a schematic of the layout of an immunosensor cartridge with anintegrated sample isolation unit in accordance with one embodiment ofthe present invention;

FIG. 6 is a flow chart of the fluid and air paths within an immunosensorcartridge with an integrated sample isolation unit in accordance withone embodiment of the present invention;

FIG. 7 illustrates a foldable cartridge housing in accordance with oneembodiment of the present invention;

FIG. 8 is a schematic of the layout of an immunosensor cartridge with anintegrated fixed sample extraction unit in accordance with oneembodiment of the present invention;

FIG. 9 is a flow chart of the fluid and air paths within an immunosensorcartridge with an integrated fixed sample extraction unit in accordancewith one embodiment of the present invention;

FIG. 10A shows a side view of one embodiment of the immunosensorcartridge of the present invention, and FIG. 10B shows enlarged detailsof the hemolysis detection device therein;

FIG. 11 illustrates the principle of operation of an electrochemicalimmunosensor;

FIG. 12 a side view of the construction of an electrochemicalimmunosensor with antibody-labeled particles (not drawn to scale); and

FIG. 13 is a top view of the mask design for the conductimetric andimmunosensor electrodes for an immunosensor cartridge in accordance withone embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to devices, e.g., single-use disposableassay cartridges, and to methods of using such devices to determine thepresence or concentration of analytes in a liquid sample. The inventionmay be particularly adapted for conducting diverse real-time or nearreal-time assays of analytes. In specific embodiments, the inventionrelates to devices that are configured for the controlled metereddilution of biological samples (e.g., blood, plasma, serum, urine,interstitial fluid and cerebrospinal fluid) and of analytes in thediluted samples using electrochemical immunosensors or otherligand/ligand receptor-based biosensors. The devices and methods alsopreferably include a feature for verifying the degree of dilution of thediluted sample, e.g., by employing a dilution determinant marker, whichmay, for example, be selected from the group consisting of anelectrochemical species, ferricyanide, ruthenium hexamine, a ferrocenemonocarboxylic acid, an optical dye, fluorescein, acridinium salt, andmethylene blue. The dilution determinant marker concentration may, forexample, be measured using an electrochemical analysis system foramperometric measurements, using a potentiostat, or using aspectrophotometer.

In a first embodiment, the devices and methods are particularly adaptedfor low range dilution of a biological sample, e.g., dilutions that areless than about 50:1 (v/v diluent:sample). In this aspect, a sample ismetered in a sample dilution chamber to form a metered sample, and thediluent (which may or may not be metered) is added to the metered sampleto form a diluted sample that may be subjected to biological analysis,e.g., in an immunoassay on one or more electrodes.

In a second embodiment, the devices and methods are particularly adaptedfor high range dilution, e.g., dilutions of about 50:1 or greater (v/vdiluent:sample), typically from 50:1 to 50,000:1. In this aspect, aportion of the sample is isolated in a fixed sample extraction unit,which preferably is formed of a wicking material. The diluent issubsequently passed over and/or through all or a portion of the fixedsample extraction unit so that it extracts a volumetrically smallportion of the sample into the diluent. The resulting highly dilutedsample may then be subjected to biological analysis. Those skilled inthe art will recognize that the exact dilution ratio at which there is atransition from the first embodiment adapted for low range dilution tothe second embodiment adapted for relatively high range dilution mayvary depending on parameters including the exact device geometry,fabrication materials and sample type.

I. Sensor Cartridge

A. Low Range Dilution Cartridge Construction

While the present invention is broadly applicable to assay systems, itis best understood in the context of the i-STAT® immunoassay system(Abbott Point of Care Inc., Princeton, N.J., USA), as described in thejointly-owned pending patent applications and issued patents citedherein.

The specific form of the devices, e.g., cartridges, of the presentinvention suitable for sample dilution may vary widely. An exemplarycartridge design according to the first (low range) dilution embodimentof the present invention is shown in FIGS. 1-6 and comprises a cover 1(FIGS. 1A and 1B), a base 3 (FIG. 3) and a thin-film adhesive gasket 21(FIG. 2) disposed between the cover 1 and the base 3. The cartridge alsoincludes a flexible, e.g., rubberized, pump membrane 9, shown in FIG. 4,which illustrates an exploded view of the cartridge. FIG. 5 illustratesa composite drawing of the exemplary cartridge superimposing thefeatures of the cover, the base and the gasket. FIG. 6 illustrates aconceptual flow diagram of the fluid and air paths within animmunosensor cartridge with an integrated sample isolation unit suitablefor low range sample dilution according to one embodiment of the presentinvention.

As shown in FIG. 1A, the cover 1 of the cartridge is made of a rigidmaterial, preferably plastic, capable of repetitive deformation withoutcracking at flexible hinge region 10. The cover 1 further comprises apaddle 7, which is moveable relative to the body of the cover 1, andwhich is attached to the body by flexible hinge region 10. A pumpopening 6 is disposed in the central region of cover 1, and a recessedpump membrane region 5 is provided, preferably on the underside of thecover, as shown in FIG. 1B, for receiving pump membrane 9. Pump membrane9 may be secured to pump membrane region 5 with an adhesive, whichshould form an air-tight seal in order to allow pump membrane 9 to berepeatably deformed during pumping operations. In other embodiments, notshown, the membrane may be secured to the outer surface of the cover 1.The underside of the cover also preferably includes various conduits andfluid flow features as shown in FIG. 1B and described below.

The base 3, shown in FIG. 3, includes a closure member 2, attached tothe main body of the base by prongs at the distal end thereof. Onenon-limiting embodiment of a closure member is described injointly-owned U.S. Pat. No. 7,682,833, the entirety of which isincorporated herein by reference. The major features of the base includepump cavity 43, diluent cavity 42, sample entry port 4, and variousconduits and fluid flow features as shown in FIG. 3 and described below.The gasket, shown in FIG. 2, is disposed between the cover and base andincludes various openings that permit fluids to pass between theconduits in the cover and conduits in the base.

In operation, a biological sample, e.g., whole blood, urine, etc., isintroduced into sample entry port 4 and preferably enters sample holdingchamber 34 (as shown in FIG. 3) passively via capillary action. Theholding chamber 34 extends from the sample entry port 4 to a capillarystop 25. As shown, capillary stop 25 is formed by a hole within thegasket (FIG. 2) that separates holding chamber 34 in the base fromanalysis conduit 15 in the cover, although in other embodiments, notshown, the capillary stop may be formed by a constriction in a conduiteither in the base or the cover of the cartridge. A capillary stop isone example of a sample isolation unit, defined herein as any devicecapable of isolating the sample in a specific conduit or region of thedevice. In another embodiment, a sample isolation unit may be formed ofa sponge or wicking material that acts to retain the sample.

After introduction of the sample, the closure member 2 can be secured,e.g., slidably secured, over the entrance of sample entry port 4 toprevent sample leakage. The cartridge is then inserted into a readingapparatus in which the sample preferably is automatically manipulated byactuators to detect the analyte in question. The cartridge is thereforepreferably adapted for insertion into a reading apparatus, and thereforehas a plurality of mechanical and electrical connections adapted forthis purpose. It should also be apparent that partial manual operationof the cartridge is possible. When operated upon by a pump means withinthe reading apparatus, pump membrane 9 exerts a force upon air within anair bladder comprised of cavity 43, which is covered by pump membrane 9,to displace fluids within conduits of the cartridge. When operated by asecond pump means, paddle 7 exerts a force upon gasket 21, which candeform because of slits 22 cut therein. (In an alternative embodiment,not shown, a second pump membrane may be substituted for paddle 7.)Gasket 21, in turn, applies pressure on a fluid-containing pouch orpackage 57, preferably a foil pack comprising a diluent fluid, that isdisposed within cavity 42. Thus, upon insertion of the cartridge intothe reading apparatus, an actuation mechanism in the reading apparatusapplies pressure to the gasket transmitting pressure onto fluid package57 filled with, for example, about 20 to 200 μL, e.g., about 160 μL ofdiluent in cavity 42, rupturing fluid package 57 upon spike 38, andexpelling diluent into conduit 39, through hole 29 in gasket 21, andinto diluent conduit 53 in the cover. Diluent is transported in diluentconduit 53 and is optionally metered in a diluent metering chamber,which is the region within conduit 53 that is between hole 48 (airintroduction port) and hole 47 (diluent introduction port). Preferably,holes 48 and 47 are small enough that the surface tension of the diluentcontained within the diluent conduit 53 inhibits or prevents the diluentfrom prematurely passing therethrough. Subsequent pumping action onmembrane 9, as described below, allows air to enter the diluent conduit53 via hole 48, and expels the diluent, preferably a metered amount ofdiluent, through hole 47 and into sample dilution chamber 52. Thediluent then passively mixes with the sample in dilution chamber 52 asthe resulting sample/diluent mixture is expelled through capillary stop25 with continued pumping action and into analysis conduit 15.

In preferred embodiments, the diluent also functions as a wash fluid andmay be separately transported to one or more electrodes in the cartridgein order to wash unbound species (e.g., unbound analyte and signalantibodies) from the electrode region after sandwich formation. In theembodiment shown in FIG. 5, diluent conduit 53 is connected to washconduit 20 in order to effect transport of the diluent, acting as washfluid, to conduit 20 and ultimately to the electrodes in analysisconduit 15 for washing purposes. As shown, the wash conduit 20 isconnected to the analysis conduit 15 via intervening conduit 8 as shownin FIG. 5. The length and orientation of conduits 20, 8 and 15 anddilution chamber 52 preferably are designed such that the diluent mixeswith the sample and passes the resulting sample/diluent mixture over theelectrodes for sandwich formation prior to directing of the separatediluent stream, acting as wash fluid, to the electrodes to wash unboundspecies therefrom.

In some embodiments, not shown, the analyzer mechanism applied to thecartridge may be used to inject one or more air segments into thediluent derived from conduit 20 (when the diluent is acting as washfluid) at controlled positions within the analysis conduit. Thesesegments may be used to help wash the sensor surface and the surroundingconduit using a minimum of fluid. The cover, for example, may furthercomprise a hole covered by a thin pliable film for this purpose. Inoperation, pressure exerted upon the film expels one or more airsegments into conduit 20 through a small hole 28 in the gasket. See, forexample, U.S. Pat. No. 7,723,099, the entirety of which is incorporatedherein by reference.

Referring to FIG. 1B, the lower surface of the cartridge cover furthercomprises a wash conduit 11, an analysis conduit 15 and a diluentconduit 53. Optional coatings within one or more of these conduits mayprovide hydrophobic surfaces, which may assist in controlling fluid flowbetween conduits 11, 20 and 15. A recess 40 in the base provides apathway for air to pass from the pump cavity 43 to hole 48 in thegasket, into conduit 53 in the cover, through hole 47 (diluentintroduction port) in the gasket, and into sample dilution chamber 52(within sample chamber 34) in the base. In operation, diluent containedin conduit 53 in the region between hole 48 and hole 47 (diluentintroduction port) in the gasket is pushed through hole 47 and intodilution chamber 52 (a region within sample chamber 34), which containsa metered sample. In this manner, the diluent mixes with the meteredsample as the two components are simultaneously pushed through thecapillary stop 25 (or other sample isolation unit) and into analysisconduit 15 for sandwich formation and analysis.

As shown in FIG. 2, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Hole 122 permits fluid to flow into sample entry port 4 andinto sample holding chamber 34. Hole 24 permits fluid to flow fromconduit 11 into waste chamber 44. Capillary stop 25 comprises an openingbetween sample dilution chamber 52 and analysis conduit 15. Hole 28permits fluid to flow from conduit 19 to waste chamber 44 via optionalcloseable valve 41. Holes 30 and 33 permit the plurality of electrodesthat are housed within cutaways 35 and 37, respectively, to contactfluid within analysis conduit 15. In a specific embodiment, cutaway 37houses a ground electrode, and/or a counter-reference electrode, andcutaway 35 houses at least one analyte sensor and, optionally, aconductimetric sensor. It should be noted that although the conduitsdescribed in connection with the figures variously traverse the gasket,in other embodiments, the conduits may be oriented substantially in thesame plane without traversing the gasket, or may traverse the gasket ina manner different than shown in FIGS. 1-5.

Referring to FIG. 3, sample holding chamber 34 extends from the sampleentry port 4 to capillary stop 25. Sample dilution chamber 52 (FIG. 5)is disposed within sample chamber 34, specifically between hole 47 andcapillary stop 25. As shown, the base includes a vent 49 thatfacilitates loading of the dilution conduit 53. Specifically, as diluentis allowed to passively enter diluent conduit 53, air that was containedin the diluent conduit is allowed to exit therefrom via hole 31 in thegasket, which is disposed over vent 49. The portion of the samplebetween hole 47 (diluent introduction port) and the capillary stop 25(or other sample isolation unit) defines a metered volume of sample fordilution. In exemplary embodiments, the metered sample (prior todilution) has a volume of from 0.5 μL to 5 μL, e.g., from 0.1 μL to 10μL or from 0.05 μL to 20 μL.

In accordance with the above description, in one embodiment, theinvention is directed to a sample metering device, comprising a sampleholding chamber oriented between a sample entry port and a sampleisolation unit and having a diluent introduction port disposedtherebetween for introduction of a diluent into the sample holdingchamber. In this embodiment, the volume within the sample holdingchamber between the diluent introduction port and the sample isolationunit defines a metered volume of a sample for analysis.

In other embodiments of the invention, not shown, multiplefluid-containing packages are utilized. In some such embodiments, eachfluid-containing package contains a different fluid, e.g., diluent, awash fluid, and/or one or more reagent fluids. An air sac or bladder iscomprised of recess 43, which is sealed on its upper surface by pumpmembrane 9. The air bladder is one embodiment of a pump means, and isactuated by pressure applied to membrane 9, which displaces air inconduit 40 and thereby displaces the diluent from diluent conduit 53(optionally metered in a diluent metering chamber) and into sampledilution chamber 52, where sample dilution occurs, and ultimatelydisplacing the diluted sample through capillary stop 25 and intoanalysis conduit 15. Other types of pumps suitable for use in thepresent invention include, but are not limited to a flexible diaphragm,a piston and cylinder, an electrodynamic pump, and a sonic pump.

The region between which diluent enters the sample dilution chamber(e.g., gasket hole 47) from conduit 53 and the capillary stop 25together define a predetermined or metered volume of the sample dilutionchamber. An amount of the sample corresponding to this volume togetherwith diluent from diluent conduit 53 are displaced into the analysisconduit 15 when the air bladder or pump is depressed. This arrangementis, therefore, one embodiment of a metering means for delivering ametered amount of an originally unmetered sample into the conduits ofthe cartridge.

Metering may be advantageous, for example, if quantitation of theanalyte is required. In other embodiments, for example when determiningthe mere presence of an analyte, metering is not necessary. A wastechamber 44 is provided for sample and/or fluid that is expelled from theconduit to prevent contamination of the outside surfaces of thecartridge. A vent 45 connecting the waste chamber 44 to the externalatmosphere is also provided to facilitate fluid entry into waste chamber44. A feature of the cartridge of one embodiment of the presentinvention is that once a sample is loaded, analysis can be completed andthe cartridge discarded without the operator or others contacting thesample.

In some embodiments of the invention, a closeable valve is providedbetween the analysis conduit and the waste chamber. See, for example,the materials described in jointly-owned U.S. Pat. No. 7,419,821, whichis referenced above and hereby incorporated by reference in itsentirety. In one embodiment, the valve is comprised of a dried spongematerial that is coated with an impermeable substance. In operation,contacting the sponge material with the sample or another fluid resultsin swelling of the sponge to fill cavity, thereby substantially blockingfurther flow of liquid into the waste chamber. The wetted valve alsoblocks the flow of air between the analysis conduit and the wastechamber, which permits the first pump means connected to the sampledilution chamber to displace fluid within the wash conduit, and todisplace fluid from the wash conduit into the analysis conduit in thefollowing manner. After the sample is exposed to the sensor for acontrolled time, the sample is moved into the post-analytical conduitwhere it can be amended with a reagent. The sample can then be movedback to the sensor and a second reaction period can be initiated.Alternatively, the post-analysis conduit can serve simply to separatethe sample segment from the sensor. Within this post-analysis conduit isa single closeable valve, which connects the air vent of the analysisconduit to the diaphragm air pump. When this valve closes, the sample islocked in the post analytical conduit and cannot be moved back to thesensor chip. There are several different design examples for this valvethat are encompassed within the present invention. Some designs areactivated mechanically, while others activate upon contact with aliquid. Other types of closeable valves that are encompassed by thepresent invention include, but are not limited to, a flexible flap heldin an open position by a soluble glue or a gelling polymer thatdissolves or swells upon contact with a fluid or sample thus causing theflap to close, and alternatively, in one specific embodiment, a thinlayer of a porous paper or similar material interposed between a conduitand either the waste chamber or ambient air such that the paper ispermeable to air while dry, but impermeable when wet. In the lattercase, it is not necessary that the closeable valve be interposed betweena conduit and the waste chamber, as the valve passes little to no liquidbefore closing. Rather, the valve is appropriately placed whenpositioned between a conduit and the ambient air surrounding thecartridge. In practical construction, a piece of filter paper is placedon an opening in the tape gasket in the fluid path to be controlled. Aircan readily move through this media to allow fluid to be moved throughthe fluid path. When the fluid is pushed over this filter, the filtermedia becomes filled with liquid and further motion through the fluidpath is stopped. As the filter becomes filled, increasing pressure isrequired to move liquid through the pores of the filter. Air flowthrough the filter is also minimized or prevented. This valve embodimentrequires very little liquid to actuate the valve, and actuation occursrapidly and reliably. Valve materials, dimensions, porosity,wettability, swelling characteristics and related parameters areselected to provide for rapid closure, within one second or more slowly,e.g., up to 60 seconds, after first contact of the valve with thesample. In certain embodiments of the invention, the closeable valve isa mechanical valve. In one embodiment, a latex diaphragm is placed inthe bottom of the air bladder on top of a specially-constructed well.The well contains two openings that fluidically connect the air vent tothe sample conduit. As the analyzer plunger pushes to the bottom of theair bladder, it presses on the latex diaphragm, which isadhesive-backed, and seals the connection between the two holes. Thisblocks the sample air vent and locks the sample in place.

FIG. 6 is a schematic view of the fluidics within an immunosensorcartridge in accordance with one embodiment of the present invention.Regions R1-R8 represent specific immunosensor cartridge components andC1-C6 represent the fluidic connections between the components. W1represents a vent, e.g., a wicking vent, which facilitates fluidmovement of diluent from R4 to R3. In particular, R1 is the sample entryport and associated components for transporting the sample to W1; R2 isthe pump (e.g., air bladder) used to displace the diluent from thediluent conduit (optionally diluent metering chamber) to a meteredvolume of sample for dilution; R3 represents the sample dilutionchamber, which terminates in a sample isolation unit IU (e.g., capillarystop); R4 is the diluent conduit (optionally comprising a diluentmetering chamber); R5 represents a diluent package and may include adiluent metering chamber; and R6 represents an optional reagent package.R6 represents an optional holding chamber for a reagent or wash fluid,which may, for example, pass to conduit 20. The conduit 20, in certainembodiments, can be coated with a reagent which dissolves into the fluidreleased from R5 and/or R6. The reagent amends the fluid so that it canbetter serve as a wash/analysis fluid in embodiments where the presenceof this reagent in the dilution fluid in R4 would not be ideal for theassay. In an alternative embodiment of the invention, C2 can optionallydirectly link R4 with R6, such as, for example via a T junction. R7represents the analysis conduit and corresponding electrodes; and R8 isa waste chamber.

As shown in FIG. 7, in some embodiments of the present invention, theimmunosensor cartridge is adapted to a foldable cartridge design of thetype described in jointly-owned U.S. Pat. Appln. No. 61/288,189,entitled “Foldable Cartridge Housings for Sample Analysis,” filed Dec.18, 2009, the entirety of which are incorporated herein by reference.

Although the use of capillary stops in a sample metering chamber areknown, the use of a sample isolation unit that comprises a porousmaterial (e.g., a porous hydrophilic or hydrophobic material, acellulose material, nitrocellulose, cotton fiber, paper, glass-filledpaper, a wicking material, a matrix material or other porous material)to form a metered sample is heretofore unknown.

Thus, in one embodiment, the invention is to a sample metering device,comprising a housing comprising a sample chamber located between asample entry port and a sample isolation unit, wherein the volumebetween the entry port and the sample isolation unit defines a meteredvolume of a sample for analysis, and wherein the sample isolation unitcomprises a porous material. Although metered samples thus formed may beused in the dilution embodiments of the invention, the use of sampleisolation units that comprise a porous material is not limited to thedilution embodiments discussed herein and may be adopted in immunoassaydevices and methods that do not form diluted samples.

B. High Range Dilution Cartridge Construction

In the second dilution embodiment of the invention, a sample is dilutedat a high dilution ratio, e.g., greater than about 50:1. The specificform of devices, e.g., cartridges, according to this embodiment of theinvention may vary widely. An exemplary cartridge design according tothe second dilution embodiment (high range dilution) of the presentinvention is shown in FIGS. 8 and 9. FIG. 8 illustrates a compositedrawing of an exemplary cartridge superimposing the features of thecover, the base and the gasket according to one embodiment of theinvention, and FIG. 9 provides a flow diagram of the fluid and air pathswithin an immunosensor cartridge with an integrated fixed sampleextraction unit in accordance with one embodiment of the presentinvention. In various embodiments, dilutions in the range of about 50:1to about 50,000:1, e.g., from 100:1 to 1,000:1 or from 5,000:1 to25,000:1, can be performed.

In many respects, the devices and methods for the high range dilutionembodiments are similar to the low range dilution embodiments. Theprimary differences will be highlighted herein, but otherwise thecartridges preferably are substantially as described above in connectionwith the low range dilution embodiments. For example, in the high rangedilution embodiments, a much smaller amount of sample is metered than inthe low range dilution embodiments. In addition, the metering itself isperformed in a different manner. In the high range dilution embodiments,the metering is conducted at the fixed sample extraction unit,preferably the distal end thereof, instead of a metered sample that isformed between two openings (47 & 25 of FIG. 5) that define the sampledilution chamber (52). Further, in the high range dilution embodiments,diluent passes over and/or through all or a portion of the fixed sampleextraction unit causing the sample contained therein to be extractedinto the diluent and thereby forming a highly diluted sample.

As a specific example, high range dilution may be conducted using a“wick wash” or fixed extraction process design. In this embodiment ofthe present invention, sample is introduced into the device and isallowed to flow, e.g., passively flow through capillary action, until itreaches a fixed sample extraction unit. After contacting the fluidsample, the extraction unit preferably becomes saturated with the sampleand inhibits or prevents further flow of the sample. A distal portion ofthe extraction unit preferably defines a metered volume of a sample fordilution. In this embodiment, a very small reproducible amount of sample(e.g., 100 pL to 2 μL, from 2 nL to 1 μL or from 50 nL to 0.5 μL) isextracted from the fixed sample extraction unit in order to form adiluted sample.

As shown in FIG. 8, the immunosensor cartridge includes a sample entryport 4, a sample introduction chamber 68 and a fixed sample extractionunit 58. At the distal end of the extraction unit 58 is a sampledilution chamber 61 where formation of the diluted sample primarilyoccurs. As shown, the sample introduction chamber 68 is oriented in thebase and the sample dilution chamber is positioned in the cover of thecartridge, although in other embodiments both chambers may be orientedin the cover or in the base. The dilution chamber 61 preferably isoriented proximate, i.e., adjacent, the fixed sample extraction unit 58such that as sample is extracted from the fixed sample extraction unit58 by diluent, it passes to the sample dilution chamber for passivemixing 61. As shown, the fixed sample extraction unit 58 is oriented inthe base. In other embodiments, not shown, the fixed sample extractionunit 58 is oriented in the cover or in both the base and the cover(extending therebetween). Sample extraction unit 58 is loaded withsample and defines a metered volume of sample for dilution. A vent 60(e.g., vent wick) may be provided adjacent the fixed sample extractionunit 58 to facilitate gas removal as the extraction unit 58 is loadedwith sample.

An analysis conduit 62 (substantially similar to the analysis conduit 15in the low range dilution embodiment) comprising an analyte-responsivesurface and positioned in the cover of the cartridge is fluidlyconnected to sample dilution chamber 61. The immunosensor cartridgefurther includes a wash conduit 63 for retaining diluent used to wash ortreat the sensor after sandwich formation. The wash conduit 63 isconnected to the analysis conduit 62 via intervening conduit 8 in thebase as shown in FIG. 8. A diluent conduit 59 is connected to the washconduit 63. Diluent is released from fluid package 57 upon rupturing ofthe package on spike 38. The released diluent is then transferred viaconduit 39, through hole 29 in the gasket, and into diluent conduit 59.In some embodiments, not shown, the fluid package includes multipleindividual fluid packages (e.g., rupturable foil pouches). In thisaspect, each individual fluid package may contain a different fluidcomposition, while in other embodiments, one or more of the multiplepouches may contain the same fluid composition. Each package may haveits own associated pumping mechanism or may share a pumping mechanisms.

In operation, after the sample has saturated the fixed sample extractionunit 58, the diluent package 57 is ruptured allowing diluent to flowinto diluent conduit 59 as discussed above. The diluent flows in diluentconduit 59 until it reaches membrane opening 65 (diluent introductionport), which optionally comprises a capillary stop. At this point, pumpmembrane 9 is actuated causing air to be delivered through conduit 64and into diluent conduit 59 via hole 66. The air entering the diluentconduit causes diluent contained in diluent conduit 59, specificallybetween hole 66 and opening 65 (e.g., a metered diluent), to passthrough opening 65 (diluent introduction port) and into contact withfixed sample extraction unit 58. Upon contact with the extraction unit58, the diluent acts to extract a small volume of sample therefrom, andthe resulting diluted sample, preferably a highly-diluted sample, isallowed to pass into sample dilution chamber 61. The diluted sample thenpasses to analysis conduit 62. As will be appreciated, the desireddilution ratio may be obtained by controlling where the dilution conduitcontacts the fixed sample extraction unit and by controlling the volumeof the diluent, e.g., metered diluent in the diluent chamber.Preferably, the portion that is washed or extracted from fixed sampleextraction unit 58 is particularly small, e.g., less than 2 vol. %, lessthan 0.2 vol. %, or less than 0.01 vol. %, of the total sample containedin the fixed sample extraction unit 58 prior to contact with thediluent.

In addition to diluting the sample, the diluent preferably functions asa wash fluid as in the low range dilution embodiment discussed above. Inthese embodiments, upon rupture of diluent package 57, some diluent isallowed to flow through hole 29 and into wash conduit 63 (wash conduit63 is shown in fluid communication with diluent conduit 59). Washconduit 63 is in fluid communication with analysis conduit 62 viaintervening conduit 8, in order to allow the diluent, acting as a washfluid, to wash any unbound components from the region of the electrodes.

In certain embodiments of the present invention, the invention is to afixed sample extraction process for high-range dilutions comprising:loading a fixed sample extraction unit with a sample, wherein the fixedsample extraction unit is proximate to a sample dilution chamber;washing (e.g., extracting) a portion of the sample from the extractionunit using a diluent, preferably a metered volume of diluent, from adiluent conduit to form a diluted sample; transporting the dilutedsample to a sensor; and performing an analyte assay at the sensor.

In another embodiment, the fixed sample extraction process furthercomprises adding a dilution determinant marker to the sample; measuringthe dilution determinant marker concentration in the sample prior tointroducing said sample into the sample dilution chamber; measuring thedilution determinant marker concentration in a portion of the samplewashed from the extraction unit; comparing the dilution determinantmarker concentration in the sample prior to introducing said sample intothe sample dilution chamber with the dilution determinant markerconcentration in the portion of the sample washed from the extractionunit; and calculating the dilution ratio. In still other embodiments,the fixed sample extraction process includes a step of adding a dilutiondeterminant marker to the extraction unit; measuring the dilutiondeterminant marker concentration in a portion of the sample washed fromthe extraction unit; and calculating the dilution ratio.

FIG. 9 is a schematic view of the fluidics within an immunosensorcartridge with an integrated fixed sample extraction unit in accordancewith one embodiment of the present invention. Regions R1, R2 and R4-R8represent specific immunosensor cartridge components, C1-C6 representthe fluidic connections between the components and W1 represents thecontrolled dilution device for high-range dilutions (e.g., a fixedsample extraction unit). In particular, R1 is the sample introductionchamber; R2 is the pump (e.g., air bladder) used to displace the diluentfrom the diluent conduit R4 (e.g., diluent metering chamber) to ametered volume of sample for dilution; R5 is the diluent package; R6represents an optional reagent package; R7 comprises the analysisconduit; and R8 is a waste chamber. In an alternative embodiment, C2 canoptionally directly link R6 with R4, such as, for example via a Tjunction.

C. Composition of the Sample Isolation Unit and Fixed Sample ExtractionUnit

In accordance with certain aspects of the present invention, thematerials that form the fixed sample extraction unit or the sampleisolation unit in the low range dilution embodiments preferably areselected to serve as an effective fluid transport mechanism. Exemplarymaterials include any material that may be suitably configured toexhibit acceptable transport kinetics. To ensure reliable extraction ofanalyte from the sample isolation unit or fixed sample extraction unit,hydrophilic materials or coatings are preferably employed. In someembodiments, the entire matrix is comprised of a hydrophilic material,while in other embodiments, only the conduit contact edge and theconduit walls are so comprised. Exemplary materials for the fixed sampleextraction unit or sample isolation unit include cellulose,nitrocellulose, cotton fiber, paper and glass-filled paper (e.g.,Leukosorb®, Pall Corporation, Port Washington, N.Y., USA). In otherembodiments of the invention, this area of the immunosensing device maybe corona treated during assembly to promote hydrophilicity. Use of ahydrophilic material ensures that bubbles formed in the diluent are nottrapped on the sample isolation unit or fixed sample extraction unit,thereby impeding analyte transfer. In some embodiments, particularly inthe low range dilution embodiments, as discussed above, the sampleisolation unit may comprise a capillary stop and may not comprise porousor matrix-type material.

In embodiments of the invention directed to high-range dilutions,suitable materials may be less porous (e.g., having a porosity of from20 μm to 0.1 μm, e.g., from 10 μm to 0.2 μm or from 5 μm to 0.5 μm, asdetermined by microscopy (e.g., visible or scanning electronmicroscopy)), than those materials suitable for relatively smallerdilutions, the effect of which is that the sample takes longer to movethrough the matrix of the fixed sample extraction unit to the extractionface. In addition, in some embodiments of the invention, materialsgenerally considered as transverse filter materials (e.g., 0.2 μm waterpurification filters with a porous outer coating, porous glasses such asVycor®, (Corning Incorporated, Corning, N.Y., USA), treated lateral flowmaterials from American Filtrona Co. (Richmond, Va., USA), filter mediafrom Millipore Corporation (Billerica, Mass., USA), filter media fromWhatman® Schleicher & Schuell® (Maidstone, Kent, UK, and others) may beused in a lateral mode as the extraction unit material. In these highdilution embodiments, a small pore volume relative to a comparativelylarge volume of diluent is advantageous (e.g., extraction unitdimensions (length, width, height) of 2 mm×2 mm×100 μm versus 100 μL ofdiluent) for controlled dilution. In this aspect, only a sample within afew hundred microns or less is extracted from the edge of the filterinto the diluent as the diluent is washed over the fixed sampleextraction unit. Exemplary volumes of sample that are incorporated intothe diluted sample may range from 50 nL to 0.5 μL, from 2 nL to 1 μL orfrom 100 pL to 2 μL.

The porosity of the isolation unit or extraction unit may be selected topreferentially trap or retard the movement of white and red blood cells.See, for example, the materials described in jointly-owned U.S. Pat. No.5,416,026, which is hereby incorporated by reference in its entirety. Assuch, in certain embodiments, the sample that is diluted in the dilutionstep may be a plasma fraction rather than whole blood. In suchembodiments, the isolation unit or extraction unit is formatted as alateral flow element where blood enters on one side and plasmapredominates towards the other side. This configuration is shown inFIGS. 10A and 10B. In the device of FIG. 10B, a hemolysis detectiondevice sample orifice 101 for contacting the whole blood sample with dryseparation material 102 is located proximate to the blood entry port 4(FIG. 8). The plasma or serum fraction wicks along dry separationmaterial 102, thus becoming separated from whole blood cells. In otherembodiments, the isolation unit or extraction unit also includes alysing agent that lyses only the red blood cells in the sample, or boththe red and the white blood cells. Suitable lysing agents may, in someembodiments, be dry coated onto the isolation unit or extraction unitfor dissolution into the sample. Preferred lysing agents include sodiumdeoxycholate and saponin.

D. Dilution Verification

In accordance with various embodiments of the present invention, theeffective dilution ratio and reproducibility of any given cartridgedesign can be ascertained or confirmed by adding one or more dilutiondeterminant markers to the sample prior to introduction to theimmunosensing device or within the device itself. In some embodiments, ameasurable concentration of ferricyanide, e.g., from 0.01 to 50, from0.1 to 10 or from 1 to 5 mM ferricyanide, is added to the sample. Otherelectrochemical species suitable for use as the dilution determinantmarker include ruthenium hexamine and a ferrocene, e.g. ferrocenemonocarboxylic acid. Alternative ways of verifying sample dilutioninclude the use of an optical dye dilution determinant marker (e.g.,fluorescein, an acridinium salt, and methylene blue). In theseembodiments, a spectrophotometer may be used to determine the ratio ofconcentrations and hence, the dilution factor. In still otherembodiments, dilution can be confirmed using a sodium ion concentrationdilution, e.g., with an integrated sodium ion sensor with the initialsample sodium concentration verified in a second cartridge.

Thus, in one embodiment, the invention is to a method of performing anassay for an analyte in a fluid sample with a cartridge having anintegrated sample dilution element, where the cartridge is adapted forinsertion into a reading apparatus, the method comprising: (a)introducing a fluid sample into a sample holding chamber of a cartridgewith a sample dilution element, wherein at least a portion of saiddilution element determines a volume of sample for dilution, and whereinsaid dilution element further comprises a predetermined known amount ofa dilution determinant marker capable of dissolving into said sample;(b) pumping a metered volume of diluent from a diluent chamber in saidcartridge, to said sample dilution element to form a diluted sample; (c)pumping the diluted sample to a sensor in a sensing region of saidcartridge; (d) measuring the concentration of said dilution determinantmarker in said diluted sample; and (e) determining the dilution ratio ofthe diluted sample from said measured concentration in step (d) and saidpredetermined known amount of step (a). The diluted sample may be at adilution ratio of from about 1:1 to 50:1 parts by volume diluent:sample,or from 50:1 to about 50,000:1 parts by volume diluent:sample. Thedilution ratio value determined in step (e) preferably is used tocalculate the concentration of the analyte in the undiluted sample.

In this embodiment, the predetermined known amount of dilutiondeterminant marker may, for example, be an embedded value in saidreading apparatus, e.g. a value or coefficient programmed into theinstrument software algorithm, an embedded value on said assay cartridgeand automatically read by said reading apparatus, e.g. a barcode, 2Dbarcode, magnetic strip, a visible value, e.g. printed number or lettercode, on said assay cartridge and manually entered into said readingapparatus, or a visible value, e.g. printed number or letter code, onsaid assay cartridge package and manually entered into said readingapparatus.

In one embodiment of the invention, a dry reagent, e.g., ferrocenemonocarboxylic acid, is added to the portion of the sample dilutionchamber and parameters are set to give a known dissolved concentrationof ferrocene in the blood sample, e.g., 0.1 to 10 mM or about 1.0 mM. Incertain embodiments, one or more sensors in the cartridge can be used todetect the ferrocene signal for the diluted sample during the sandwichformation step. For an intended hundred fold dilution, for example, thesignal may be equivalent to 10 μM ferrocene. During factory calibration,a current versus concentration algorithm can be established and embeddedin the instrument software. Without being bound by theory, as ferrocenegives an outer sphere electron transfer reaction, the signal should notdepend on the electrode catalytic activity. Ferrocene monocarboxylicacid has a half-wave potential about 200 mV more positive than thep-aminophenol (PAP) used to detect the sandwich, so it should notprovide a material signal, even if the wash step leaves residue.

In another embodiment, the sample is introduced to the device and passesonto the sample isolation unit or, in other embodiments of theinvention, to the fixed sample extraction unit. When the dilutionelements are actuated and the diluted sample is passed to the detectionregion of the immunosensing device, in certain embodiments of theinvention, a portion of the diluted sample may be manually removed andtested using a potentiostat or other electrochemical analysis system foramperometric measurements, a potentiometer for potentiometricelectrochemical tests, or a spectrophotometer to determine the dilutiondeterminant marker concentration. The ratio of the dilution determinantmarker concentration in the undiluted sample to the dilution determinantmarker concentration in the analyzed portion of the diluted sampleprovides the dilution ratio for any given design, and by repeating thecharacterization for a set of devices, the precision and accuracy of thedilution process may be calculated. As such, embodiments of the presentinvention provide for independent empirical verification of accuracy andprecision of dilution system designs. Use of the dilution determinantmarker may also serve as a control test during actual use of theimmunosensing device of the present invention.

While an assay may, in principle, require a defined target dilutionratio (e.g., 5,000:1), in some embodiments of the present invention, itmay be found that a particular sample isolation unit or fixed sampleextraction unit dilution has high precision but is inaccurate (e.g.,gives a ratio of 5,300:1). In certain embodiments, the assaycoefficients are adjusted (in this example, to a 5,300:1 ratio) ratherthan reengineer the design elements. Those skilled in the art willrecognize that for practical assay development using a single-usecartridge format, this is one viable approach. Note that while thepresent disclosure uses integer ratios for convenience, fractionalratios, e.g. 2:7, 2:7.1, 2:7.01, are also anticipated to be within thescope of the invention.

In preferred embodiments of the invention, the diluent fluid is astimulant of plasma without the presence of the analyte. In certainembodiments, the diluent fluid is aqueous based and includeselectrolytes, buffers and proteins typically found in plasma at highconcentration, e.g., albumin and immunoglobulins. The diluent fluid mayalso include lysing agents, stabilizers, and antibacterial agents, whichare well-known in the clinical biochemical arts.

With regard to the transit time of the diluent fluid in contact with thecontrolled dilution device (e.g., time during washing or the extractionof a portion of the analyte out of the controlled dilution device), thediluent fluid volume will generally be selected in the range of about 5μL to about 200 μL. In some embodiments, the transit time across thesurface (e.g., face or edge) of the controlled dilution device may be inthe range of about 0.1 second to about 100 seconds (e.g., 1 second to 50seconds or 2 seconds to 10 seconds).

In certain embodiments, the diluent fluid is transported through theconduit and across the surface of the controlled dilution device at asubstantially fixed flow rate, e.g. 10 μL/s. The quicker the diluentfluid moves, the less time is provided for extraction of analyte fromthe dilution device. As such, rate of fluid flow is a control parameterfor the assay system. In some embodiments, the fluid flow can becontrolled by an instrument mechanism and software, which controlactuation of the pump elements. Alternative embodiments to control offlow rate include a pump cycle that has a fixed stationary dwell timefor a portion of the diluent fluid in contact with the surface of thecontrolled dilution device, and also a pump cycle that oscillates aportion of the diluent across the surface of the controlled dilutiondevice. In these embodiments, one or more software programs can beutilized to control the instrument mechanism interaction with the pumpelements.

With regard to the fixed sample extraction unit for high levels ofdilution, in some embodiments, instrument software includes a delayfeature such that the instrument does not deploy the diluent untilsufficient time has elapsed. In a preferred embodiment, the instrumentincludes a detector switch that registers the time of insertion to thetest device, and this acts as the t=0 point for the test cycle. Thus, ifthe fixed sample extraction unit filling step takes 15 seconds from thetime the sample enters the device, diluent activation is set to beinitiated at t>15 (e.g., t=20 seconds, t=30 seconds or t=60 seconds).

II. Methods of Performing Assays

The present invention is applicable to methods of performing assays witha sensor cartridge incorporating an integrated sample dilution featureand sample metering device. The methods of the invention are applicableto various biological sample types (e.g., blood, plasma, serum, urine,interstitial fluid and cerebrospinal fluid).

In some embodiments, the sensor cartridge is an ion sensor (e.g.,potentiometric sensor for K, Na, Cl, Ca, NH₄ and the like); a metabolitesensor (e.g., amperometic enzymatic sensor for glucose, creatinine,cholesterol and the like); an enzyme activity sensor (e.g., for livertests including ALT and AST); and a nucleotide sensor (e.g., whereamplified target ssDNA forms a sandwich with ssDNA immobilized on thesensor and other complimentary ssDNA labeled with a signal moiety suchas an enzyme or fluorescent species).

In preferred embodiments, the present invention may be employed in oneor more of the following areas: immunosensors, most notably in thecontext of point-of-care testing; electrochemical immunoassays;immunosensors in conjunction with immuno-reference sensors; whole bloodimmunoassays; single-use cartridge based immunoassays; andnon-sequential immunoassays with only a single wash step; and dryreagent coatings. As will be appreciated by those skilled in the art,the general concept disclosed herein is applicable to many immunoassaymethods and platforms. In addition, the present invention is applicablea variety of immunoassays, including both sandwich and competitiveimmunoassays.

After controlled sample dilution and sandwich formation on animmunosensor, in accordance with various embodiments of the invention,wash or diluent is deployed. The diluent is preferably advanced throughthe connecting conduit and across the sensors by a sequence of smalldisplacement steps formed by alternating air and fluid segments. In someembodiments, segment formation is achieved by the instrument applying adisplacement force to actuators in contact with the air bladder anddiluent package in an alternating sequence. This process effectivelyentrains a set of air and fluid segments over the sensor. It has beenfound that the meniscus between each segment is the most effective partof the wash cycle to remove sample, unbound analyte and unbound signalantibody from the sandwich formation or sensor region of the cartridge.In addition, a sequence of segments provides a more complete wash of thesensor compared to the same volume of diluent applied in a single passover the sensor, although the latter may be used in assays wherenon-specific binding is not a significant issue. In preferredembodiments, the air and fluid segments each have a volume of about 2μL, but the segment volume can range from less than 1 μL to more than 20μL. In certain embodiments of the invention where the analysis conduithas a cross-sectional area of about 1-2 mm³, each fluid segment isseparated by an air gap of about 2-3 mm. In addition, in someembodiments, a conductivity sensor may be positioned in the analysisconduit to monitor the position of fluid-air interfaces and providefeedback control to the instrument software for pump actuation. (See,for example, the materials described in jointly-owned U.S. Pat. No.7,419,821, which is referenced above and hereby incorporated byreference in its entirety.)

In alternative embodiments, a segment is injected using a passivefeature. A well in the base of the cartridge is sealed by a tape gasket.The tape gasket covering the well has two small holes on either end. Onehole is open while the other is covered with a filter material that wetsupon contact with a fluid. The well is filled with a loose hydrophilicmaterial such as, for example, a cellulose fiber, paper or glass fiber.The hydrophilic material draws the liquid into the well in the base viacapillary action, displacing the air that was formerly in the well. Theair is expelled through the opening in the tape gasket, creating asegment whose volume is determined by the volume of the well and thevolume of the loose hydrophilic material. The material used to cover oneof the inlets to the well in the base can be chosen to meter the rate atwhich the fluid fills the well and thereby control the rate at which thesegment is injected into the conduit in the cover. This passive featurepermits any number of controlled segments to be injected at specificlocations within a fluid path and requires a minimum of space.

Within a segment of sample or fluid, an amending substance can bepreferentially dissolved and concentrated within a predetermined regionof the segment. This is achieved through control of the position andmovement of the segment. Thus, for example, if only a portion of asegment, such as the leading edge, is reciprocated over the amendedsubstance, then a high local concentration of the substance can beachieved close to the leading edge. Alternatively, if an homogenousdistribution of the substance is desired, for example if a knownconcentration of an amending substance is required for a quantitativeanalysis, then further reciprocation of the sample or fluid will resultin mixing and an even distribution.

In various embodiments of the invention, one or more portions of thecomponents, conduits, and/or controlled dilution device can be coatedwith a dry reagent to amend a sample or fluid. The sample or fluid ispassed at least once over the dry reagent coating to dissolve it.Reagents used to amend samples or fluid within the cartridge include,but are not limited to antibody-enzyme conjugates, signal antibodies tothe target analyte, or blocking agents that prevent either specific ornon-specific binding reactions among assay compounds. A surface coatingthat is not soluble, but helps prevent non-specific adsorption of assaycomponents to the inner surfaces of the cartridges can also be utilizedin some embodiments of the present invention.

As described above, the immunosensor cartridge may further include anindividual diluent package containing a diluent and/or an individualreagent fluid package containing a reagent fluid. In certainembodiments, these packages are in the form of a rupturable pouch (e.g.,foil pouch). Manufacture of the rupturable pouches may be performed, forexample, as described in jointly-owned U.S. Pat. Appln. Pub. No.2010/0068097 A1 to Ade et al. or in jointly-owned U.S. Pat. No.5,096,669 to Lauks et al., the entirety of each of which is incorporatedherein by reference.

The composition of the diluent is preferably selected to include abuffer, pH, detergents and the like to promote removal of the unboundsample and non-specifically bound signal antibody, without substantialeffect on the stability of the sandwich formed on the immunosensor.Those skilled in the immunoassay art will recognize that such diluentcompositions are well-known, as are methods for their optimization for agiven assay format. In some embodiments of the invention, the volume ofthe diluent in the diluent package is selected to be in the range ofabout 50 μL to about 200 μL.

The composition of the detection or reagent fluid is selected to includean enzyme substrate, buffer, pH, detergents and the like to promoteefficient activity of the enzyme on the signal antibody, withoutsubstantial effect on the stability of the sandwich formed on theimmunosensor. Reagent fluid compositions are well-known in theimmunoassay art, as are methods for their optimization for a given assayformat. In some embodiments, the detection fluid in the detection fluidpackage is in the volume range of about 50 μL to about 200 μL andcontains p-aminophenol (PAP) phosphate as a substrate for the alkalinephosphatase enzyme label in a buffered solution at pH 9.8.

III. Ratiometric Immunoassays

Given the relative sensitivity of the antibodies that are used and theactual whole blood concentrations of certain protein molecules (e.g.,hemoglobin or albumin), it may be desirable to reduce their respectiveconcentrations, for example, down to the range of about 1 to 100 ng/mL,which is roughly a 500 to 5000 fold dilution. In these embodiments,accurate dilution is not critical. Rather, the sample need only bediluted down to the analyte concentration range where the sensorresponse is quasi-linear.

In certain embodiments, the sample isolation unit approach for low-rangedilutions can be utilized to perform a ratiometric assay in a bloodsample. For example, in one embodiment of the present invention, amethod of performing a ratiometric assay in a blood sample is providedcomprising introducing a blood sample into a sample dilution chamber ofa cartridge, wherein the dilution chamber is located between a diluentintroduction port and a sample isolation unit, and wherein the volumebetween the diluent introduction port and the sample isolation unitdefines a metered volume of said sample for dilution. The metered volumeof the sample is diluted with a metered volume of diluent (as describedabove) from a diluent conduit located within the cartridge to form adiluted sample. The diluted sample is pumped to a first and secondsensor in a sensing region (e.g., analysis conduit) of the cartridge,the first sensor comprising an immunosensor for a first analyte and thesecond sensor comprising an immunosensor for a second analyte. A firstsandwich is formed on the first sensor comprising an immobilized firstanalyte antibody, the first analyte and a first analyte antibody labeledwith a signaling moiety, and a second sandwich is formed on the secondsensor comprising an immobilized second analyte antibody, the secondanalyte and a second analyte antibody labeled with the signaling moiety.The diluted sample is subsequently washed from the sensing region of thecartridge, optionally with a wash fluid that is the same composition asthe diluent, and a reagent is introduced for generating a signal fromthe signaling moiety to the sensing region of the cartridge. The signalat said first and second sensors is detected and recorded, and thefractional percentage of the first analyte to the second analyte isdetermined from the signal at said first and second sensors.

In certain embodiments, the sample isolation unit approach forhigh-range dilutions can be utilized to perform a ratiometric assay in ablood sample. For example, in one embodiment, the invention is to amethod of performing a ratiometric assay in a blood sample, comprisingintroducing a blood sample into a sample introduction chamber of acartridge, wherein the introduction chamber terminates in a fixed sampleextraction unit, and wherein a distal portion of said extraction unitdefines a metered volume of a sample for dilution. The metered volume ofthe sample is diluted with a metered volume of diluent from a diluentconduit located within the cartridge to form a diluted sample, which ispumped to first and second sensors in a sensing region of the cartridge.The first sensor comprises an immunosensor for a first analyte and thesecond sensor comprises an immunosensor for a second analyte. A firstsandwich is formed on the first sensor comprising an immobilized firstanalyte antibody, the first analyte and a first analyte antibody labeledwith a signaling moiety, and a second sandwich is formed on the secondsensor comprising an immobilized second analyte antibody, the secondanalyte and a second analyte antibody labeled with the signaling moiety.The diluted sample is subsequently washed from the sensing region of thecartridge, and a reagent for generating a signal from the signalingmoiety is introduced to the sensing region of the cartridge. The signalat said first and second sensors is detected and recorded and thefractional percentage of the first analyte to the second analyte isdetermined from the signals at said first and second sensors.

In certain embodiments of the invention, the first analyte compriseshemoglobin and the second analyte comprises hemoglobin A1c. In thisembodiment, the dilution chamber or the controlled dilution devicepreferably comprises a lysing agent (e.g., sodium deoxycholate orsaponin) capable of dissolving in the sample, the diluent or the dilutedsample. In other embodiments where the first analyte comprises albuminand the second analyte comprises glycosylated albumin, a lysing agent isnot required.

EXAMPLES

The present invention will be better understood with reference to thespecific embodiments set forth in the following non-limiting propheticexamples. Suitable non-limiting examples of analytes detectable with thelow dilution format are hemoglobin A1c and C-reactive protein. Suitablenon-limiting examples of analytes detectable with high dilution formatare hemoglobin, human serum albumin and immunoglubulins, e.g., IgG andIgA. Typical disease states include anemia and immunity assessment. Inaddition, detection of beta human chorionic gonadotropin (bHCG) can beextended into the range of about 50,000 to 500,000 ng/mL.

Example 1. Amperometric Immunoassay

FIG. 11 illustrates the principle of an amperometric immunoassayaccording to specific non-limiting embodiments of the present inventionfor determination of C-reactive protein (CRP) 70 in a diluted fluidsample, a marker of inflammation. A diluted sample (e.g., whole bloodsample) according to the invention, as described above (either high orlow range dilution, but preferably the latter for CRP) is mixed with aconjugate molecule 71 comprising alkaline phosphatase enzyme (AP)covalently attached to a polyclonal anti-CRP antibody (aCRP) 72. Theconjugate 71 specifically binds to CRP 70 in the sample, producing acomplex made up of CRP 70 bound to the AP-aCRP conjugate 71. In acapture step, this complex binds to the capture aCRP antibody 72attached onto the surface of or unattached but proximate to the sensor(e.g., gold electrode 74). In some embodiments, a conductivity sensor(not shown) is used to monitor when a certain volume of the sample(e.g., sample segment) reaches the sensor. The time of arrival of thesample can be used to detect leaks within the cartridge (e.g., a delayin arrival signals a leak). The position of the sample segment withinthe analysis conduit can be actively controlled using the edge of thefluid sample as a marker. As the sample/air interface crosses theconductivity sensor, a precise signal is generated that can be used as afluid marker from which controlled fluid excursions can be executed. Thesample segment is preferentially oscillated edge-to-edge over the sensor74 in order to present the entire sample to the sensor surface. A secondreagent can be introduced in the analysis conduit beyond the sensor,which becomes homogenously distributed during the fluid oscillations.The sensor chip contains a capture region or regions coated withantibodies for the analyte of interest. These capture regions aredefined by a hydrophobic ring of polyimide or anotherphotolithographically produced layer. A microdroplet or severalmicrodroplets (5-40 nL in size) containing antibodies in some form,e.g., bound to latex microspheres, is dispensed on the surface of thesensor. The photodefined ring contains the one or more aqueous droplets,allowing the antibody coated region to be localized to a precision of afew microns. In some embodiments, the capture region can be made from0.03 mm² to 2 mm² in size. The upper end of this size range is limitedby the size of the conduit and sensor in certain embodiments, and is nota limitation of the invention.

Thus, the gold electrode 74 is coated with a biolayer 73 comprising acovalently attached anti-CRP antibody, to which the CRP/AP-aCRP complexbinds. AP is thereby immobilized close to the electrode in proportion tothe amount of CRP initially present in the sample. In addition tospecific binding, the enzyme-antibody conjugate may bindnon-specifically to the sensor. Non-specific binding provides abackground signal from the sensor that is undesirable and preferably isminimized. As described above, the rinsing protocols, and in particularthe use of segmented fluid to rinse the sensor, provide efficient meansto minimize this background signal. In a second step subsequent to therinsing step, a substrate 75 that is hydrolyzed by, for example,alkaline phosphatase to produce an electroactive product 76 is presentedto the sensor. The amperometric electrode is either clamped at a fixedelectrochemical potential sufficient to oxidize or reduce a product ofthe hydrolyzed substrate but not the substrate directly, or thepotential is swept one or more times through an appropriate range.Optionally, a second electrode may be coated with a layer where thecomplex of CRP/AP-CRP is made during manufacture, to act as a referencesensor or calibration means for the measurement.

In the present example, the sensor comprises two amperometric electrodesthat are used to detect the enzymatically produced 4-aminophenol fromthe reaction of 4-aminophenylphosphate with the enzyme label alkalinephosphatase. The electrodes are preferably produced from gold surfacescoated with a photodefined layer of polyimide. Regularly spaced openingsin the insulating polyimide layer define a grid of small gold electrodesat which the 4-aminophenol is oxidized in a 2 electron per moleculereaction.H₂N—C₆H₄—OH→HN═C₆H₄═O+2H⁺+2e ⁻

Sensor electrodes further comprise a biolayer, while referenceelectrodes can be constructed, for example, from gold electrodes lackinga biolayer, or from silver electrodes, or other suitable material.Different biolayers can provide each electrode with the ability to sensea different analyte.

Substrates, such as p-aminophenol (PAP) species, can be chosen such thatthe E_(1/2) of the substrate and product differ substantially.Preferably, the E_(1/2) of the substrate is substantially higher thanthat of the product. When the condition is met, the product can beselectively electrochemically measured in the presence of the substrate.In specific embodiments, the substrate is comprised of a phosphorylatedferrocene or, more preferably, phosphorylated PAP.

The size and spacing of the electrode play an important role indetermining the sensitivity and background signal. The importantparameters in the grid are the percentage of exposed metal and thespacing between the active electrodes. The position of the electrode canbe directly underneath the antibody capture region or offset from thecapture region by a controlled distance. The actual amperometric signalof the electrodes depends on the positioning of the sensors relative tothe antibody capture site and the motion of the fluid during theanalysis. A current at the electrode is recorded that depends upon theamount of electroactive product in the vicinity of the sensor.

The detection of alkaline phosphatase activity in this example relies ona measurement of the 4-aminophenol oxidation current. This is achievedat a potential of about +60 mV versus the Ag/AgCl ground chip. The exactform of detection used depends on the sensor configuration. In oneversion of the sensor, the array of gold microelectrodes is locateddirectly beneath the antibody capture region. When the diluent is pulledover this sensor, enzyme located on the capture site converts the4-aminophenylphosphate to 4-aminophenol in an enzyme limited reaction.The concentration of the 4-aminophenylphosphate is selected to be inexcess, e.g., 10 times the Km value. The analysis solution is 0.1 M indiethanolamine, 1.0 M NaCl, buffered to a pH of 9.8. Additionally, theanalysis solution contains 0.5 mM MgCl, which is a cofactor for theenzyme.

In another electrode geometry embodiment, the electrode is located a fewhundred microns away from the capture region. When a fresh segment ofdiluent is pulled over the capture region, the enzyme product buildswith no loss due to electrode reactions. After a time, the solution isslowly pulled from the capture region over the detector electrode,resulting in a current spike from which the enzyme activity can bedetermined.

An important consideration in the sensitive detection of alkalinephosphatase activity is the non-4-aminophenol current associated withbackground oxidations and reductions occurring at the gold sensor. Goldsensors tend to give significant oxidation currents in basic buffers atthese potentials. The background current is largely dependent on thebuffer concentration, the area of the gold electrode (exposed area),surface pretreatments and the nature of the buffer used. Diethanolamineis a particularly good activating buffer for alkaline phosphatase. Atmolar concentrations, the enzymatic rate is increased by about threetimes over a non-activating buffer such as carbonate.

In alternative embodiments, the enzyme conjugated to an antibody orother analyte-binding molecule is urease, and the substrate is urea.Ammonium ions produced by the hydrolysis of urea are detected in thisembodiment by the use of an ammonium sensitive electrode.Ammonium-specific electrodes are well-known to those of skill in theart. A suitable microfabricated ammonium ion-selective electrode isdisclosed in U.S. Pat. No. 5,200,051, which is referenced above andhereby incorporated by reference in its entirety. Other enzymes thatreact with a substrate to produce an ion are known in the art, as areother ion sensors for use therewith. For example, phosphate producedfrom an alkaline phosphatase substrate can be detected at a phosphateion-selective electrode.

Referring now to FIG. 12, there is illustrated the construction of anembodiment of a microfabricated immunosensor. Preferably, a planarnon-conducting substrate is provided 80, onto which is deposited aconducting layer 81 by conventional means or microfabrication, known tothose of skill in the art. The conducting material is preferably a noblemetal such as gold or platinum, although other unreactive metals such asiridium may also be used, as may non-metallic electrodes of graphite,conductive polymer, or other materials. An electrical connection 82 isalso provided. A biolayer 83 is deposited onto at least a portion of theelectrode. In the present disclosure, a biolayer refers to a porouslayer comprising on its surface a sufficient amount of a molecule 84that can either bind to an analyte of interest, or respond to thepresence of such analyte by producing a change that is capable ofmeasurement. Optionally, a permselective screening layer may beinterposed between the electrode and the biolayer to screenelectrochemical interferents as described in U.S. Pat. No. 5,200,051,which is referenced above and hereby incorporated by reference in itsentirety.

In some embodiments of the present invention, a biolayer is constructedfrom latex beads of specific diameter in the range of 0.001 μm to 50 μm.The beads are modified by covalent attachment of any suitable moleculeconsistent with the above definition of a biolayer. Many methods ofattachment exist in the art, including providing amine reactiveN-hydroxysuccinimide ester groups for the facile coupling of lysine orN-terminal amine groups of proteins. In specific embodiments, thebiomolecule is chosen from among ionophores, cofactors, polypeptides,proteins, glycopeptides, enzymes, immunoglobulins, antibodies, antigens,lectins, neurochemical receptors, oligonucleotides, polynucleotides,DNA, RNA, or suitable mixtures. In more specific embodiments, thebiomolecule is an antibody selected to bind one or more of humanchorionic gonadotrophin, C-reactive protein, hemoglobin, hemoglobin A1c,IgG, IgA, brain natriuretic peptide (BNP), troponin I, troponin T,troponin C, a troponin complex, creatine kinase, creatine kinase subunitM, creatine kinase subunit B, myoglobin, myosin light chain, or modifiedfragments of these. Such modified fragments are generated by oxidation,reduction, deletion, addition or modification of at least one aminoacid, including chemical modification with a natural moiety or with asynthetic moiety. Preferably, the biomolecule binds to the analytespecifically and has an affinity constant for binding analyte ligand ofabout 10⁷ to 10¹⁵M⁻¹.

In one embodiment, the biolayer, comprising beads having surfaces thatare covalently modified by a suitable molecule, is affixed to the sensorby the following method. A microdispensing needle is used to depositonto the sensor surface a small droplet, preferably about 0.4 nL, of asuspension of modified beads. The droplet is permitted to dry, whichresults in a coating of the beads on the surface that resistsdisplacement during use.

In addition to immunosensors in which the biolayer is in a fixedposition relative to an amperometric sensor, the present invention alsoenvisages embodiments in which the biolayer is coated upon particlesthat are mobile. In certain embodiments, the cartridge can containmobile microparticles capable of interacting with an analyte, forexample magnetic particles that are localized to an amperometricelectrode subsequent to a capture step, whereby magnetic forces are usedto concentrate the particles at the electrode for measurement. See, forexample, jointly-owned U.S. patent application Ser. No. 12/815,132 andU.S. Provisional Pat. Appln. Ser. Nos. 61/371,066; 61/371,109;61/371,077; and 61/371,085. Each of these patent applications is herebyincorporated by reference in its entirety. One advantage of mobilemicroparticles in the present invention is that their motion in thesample or fluid accelerates binding reactions, making the capture stepof the assay faster. For certain embodiments using non-magnetic mobilemicroparticles, a porous filter is used to trap the beads at theelectrode.

Referring now to FIG. 13, there is illustrated a mask design for severalelectrodes upon a single substrate in accordance with one embodiment ofthe present invention. By masking and etching techniques, independentelectrodes and leads can be deposited. Thus, a plurality ofimmunosensors, 94 and 96, and conductimetric sensors, 90 and 92, areprovided in a compact area at low cost, together with their respectiveconnecting pads, 91, 93, 95, and 97, for effecting electrical connectionto the reading apparatus. In principle, a very large array of sensorscan be assembled in this way, each sensitive to a different analyte oracting as a control sensor.

In specific embodiments of the present invention, immunosensors areprepared as follows. Silicon wafers are thermally oxidized to form aninsulating oxide layer having a thickness of about 1 μm. Atitanium/tungsten layer is sputtered onto the oxide layer to apreferable thickness of between 100-1000 Å, followed by a layer of goldthat is most preferably 800 Å thick. Next, a photoresist is spun ontothe wafer and is dried and baked appropriately. The surface is thenexposed using a contact mask, such as a mask corresponding to thatillustrated in FIG. 13. The latent image is developed, and the wafer isexposed to a gold-etchant. The patterned gold layer is coated with aphotodefinable polyimide, suitably baked, exposed using a contact mask,developed, cleaned in an O₂ plasma, and preferably imidized at 350° C.for about 5 hours. The surface is then printed with antibody-coatedparticles. Droplets, preferably of about 0.4 nL volume and containing 2%solid content in deionized water, are deposited onto the sensor regionand are dried in place by air drying. Optionally, an antibodystabilization reagent (e.g., StabilCoat® SurModics, Inc., Eden Prairie,Minn., USA) is overcoated onto the sensor. Drying the particles causesthem to adhere to the surface in a manner that prevents dissolution ineither sample or fluid containing a substrate. This method provides areliable and reproducible immobilization process suitable formanufacturing sensor chips in high volume.

Example 2. Immunosensing Device and Method of Use

The present example describes one of the methods of use of a cartridgeembodied in the present invention. In this embodiment, the cartridgeincludes a closeable valve, located between the immunosensor and thewaste chamber. For a CRP assay, a blood sample is first introduced intothe sample chamber of the cartridge. In the following time sequence,time zero (t=0) represents the time at which the cartridge is insertedinto the cartridge reading device. Times are given in minutes. Betweent=0 and t=1.5, the cartridge reading device makes electrical contactwith the sensors through electrical contact pads and performs certaindiagnostic tests. Insertion of the cartridge perforates the foil pouchintroducing diluent into the wash conduit, as previously described, aswell as into the diluent conduit. The diagnostic tests determine whetherfluid or sample is present in the conduits using the conductivityelectrodes, determine whether electrical short circuits are present inthe electrodes, and ensure that the sensor and ground (e.g.,reference/counter) electrodes are thermally equilibrated to, preferably,37° C. prior to the analyte determination.

Between t=0.5 and t=1.5, the pumping means pumps a metered diluent fromthe diluent conduit into a dilution chamber, where the diluent is mixedwith a metered portion of the sample to form a diluted sample.

Between t=1.5 and t=6.75, the diluted sample, preferably between about 4μL and about 200 μL, more preferably between about 4 μL and about 20 μL,and most preferably about 7 μL, is used to contact the sensor. The edgesdefining the forward and trailing edges of the diluted sample arereciprocally moved over the conductivity sensor region at a frequencythat is preferably between 0.2 to 5.0 Hz, and is most preferably 0.7 Hz.During this time, the enzyme-antibody conjugate and beads (e.g., mobilebeads or magnetically-susceptible beads) dissolve within the sample. Theamount of enzyme-antibody conjugate that is coated onto the conduit isselected to yield a concentration when dissolved that is preferablyhigher than the highest anticipated CRP concentration, and is mostpreferably six times higher than the highest anticipated CRPconcentration in the sample.

Between t=6.75 and t=10.0, the diluted sample is moved to theimmunosensor for capture of the beads. As shown in FIGS. 1-4, the sampleis moved into the waste chamber via closeable valve 41, wetting thecloseable valve and causing it to close. The seal created by the closingof the valve 41 permits the first pump means to be used to controlmotion of fluid from conduit 11 to analysis conduit 15. After the valve41 closes and the remaining sample is locked in the post analysisconduit, the analyzer plunger retracts from the flexible diaphragm ofthe pump means, creating a partial vacuum in the analysis conduit. Thisforces the diluent through the small hole in the tape gasket 21 and intoa short transecting conduit 8 in the base, and then up through anotherhole in gasket 21 and into analysis conduit 15 (in cover 1). The diluentis then pulled further and the front edge of the diluent (acting here aswash fluid) is oscillated across the surface of the immunosensor chip inorder to shear the sample near the walls of the conduit. Theconductivity sensor on the chip is used to control this process.

The efficiency of the wash is optimally further enhanced by introductioninto the fluid of one or more menisci or air segments. The air segmentsmay be introduced by either active or passive means. Fluid is thenforcibly moved towards the sensor chip by the partial vacuum generatedby reducing the mechanical pressure exerted upon pump membrane 9,causing the analysis conduit 15 in the vicinity of transecting conduit 8to fill with diluent as wash fluid. This region of the analysis conduitoptionally has a higher channel height resulting in a meniscus with asmaller radius of curvature. The region of the analysis conduit in thedirection of the one or more sensors optionally has a conduit height isthat is smaller. In one aspect, the diluent passively flows from theregion adjacent the transecting conduit 8 towards this low height regionof the analysis conduit, thereby washing the conduit walls. This passivewicking effect allows further effective washing of the analysis conduitusing a minimal volume of fluid and without displacing the beads thatare attached to the sensor. In this embodiment, the fluid located withinthe wash conduit may also contain a substrate for the enzyme. In otherembodiments, amendment of the fluid using dried substrate within thewash conduit may be utilized.

Following the positioning of a final segment of fluid over the sensor,measurement of the sensor response is recorded and the concentration ofanalyte is determined. Specifically, at least one sensor reading of asample is made by rapidly placing over the sensor a fresh portion offluid containing a substrate for the enzyme. Rapid displacement bothrinses away product previously formed, and provides a new substrate tothe electrode. Repetitive signals are averaged to produce a measurementof higher precision, and also to obtain a better statistical average ofthe baseline, represented by the current immediately followingreplacement of the solution over the immunosensor.

The invention described and disclosed herein has numerous benefits andadvantages compared to previous devices. These benefits and advantagesinclude, but are not limited to ease of use, the automation of most ifnot all steps of the analysis, which eliminates user included error inthe analysis. While the invention has been described in terms of variouspreferred embodiments, those skilled in the art will recognize thatvarious modifications, substitutions, omissions and changes can be madewithout departing from the spirit of the present invention. Accordingly,it is intended that the scope of the present invention be limited solelyby the scope of the following claims.

What is claimed is:
 1. A method of performing an assay for an analyte ina sample, said method comprising the steps of: adding a dilutiondeterminant marker to the sample; determining the dilution determinantmarker concentration in the sample; introducing the sample into a samplechamber of a cartridge, wherein the sample chamber terminates in asample extraction unit; loading said extraction unit with said sample;washing a portion of said sample from the extraction unit using a volumeof diluent from a diluent conduit to form a diluted sample; introducingsaid diluted sample to a reagent comprising a conjugate molecule;measuring the dilution determinant marker concentration in said dilutedsample; comparing the dilution determinant marker concentration in thesample prior to introducing said sample into the sample chamber with thedilution determinant marker concentration in the portion of the samplewashed from the extraction unit; and analyzing said diluted sample foran analyte.
 2. The method of claim 1, wherein the dilution determinantmarker is selected from the group consisting of an electrochemicalspecies, ferricyanide, ruthenium hexamine, a ferrocene, ferrocenemonocarboxylic acid, an optical dye, fluorescein, an acridinium salt,and methylene blue.
 3. The method of claim 1, wherein the dilutiondeterminant marker concentration is measured using an electrochemicalanalysis system for amperometric measurements.
 4. The method of claim 1,wherein the dilution determinant marker concentration is measured usinga potentiometric sensor.
 5. The method of claim 1, wherein the dilutiondeterminant marker concentration is measured using a spectrophotometer.6. The method of claim 1, wherein the dilution determinant marker is adifferent entity from that of the conjugate molecule.
 7. The method ofclaim 1, further comprising calculating a dilution ratio based on thecomparison of the dilution determinant marker concentration in thesample prior to introducing said sample into the sample chamber with thedilution determinant marker concentration in the portion of the samplewashed from the extraction unit.
 8. The method of claim 7, furthercomprising calculating a concentration of the analyte in the samplebased on the analyzing of the diluted sample and the calculated dilutionratio.